Novel glycopeptide and methods of isolating and using the same

ABSTRACT

A novel glycopeptide is disclosed, which consists of a sugar moiety having a structural formula ##STR1## and a peptide moiety linking to the sugar moiety through N-glycoside bond. The glycopeptide is isolated from organs of normal animals, especially kidney, or urine from healthy human being, and has a biological activity of inducing lesions of kidney in animals. The lesions of kidney induced by an injection of the glycopeptide are very similar to spectrum of human glomerulonephritis. Accordingly, an experimental model for study on human glomerulonephritis is established.

BACKGROUND OF THE INVENTION

The present invention relates to a novel glycopeptide consisting of asugar moiety having a structural formula ##STR2## and a peptide moietylinking to the sugar moiety through N-glycoside bond and a process ofisolating it. The present invention relates also to an agent of inducingglomerulonephritis in an animal and to a method of preparing anexperimental model for study on human glomerulonephritis.

It is very important in medical and pharmaceutical fields, especiallyfor an development of therapeutic treatment of human disease, screeningof medicines or the like, to prepare stably, plentifully andeconomically an experimental model, in other words, an experimentalanimal having lesions which are strikingly similar to spectrum of humandisease. Recently, the supply of experimental models such as, forexample, rat with hypertension, mouse with diabetes, rat with digestiveulcer or the like has contributed remarkably to the development oftherapeutic treatments.

Although, with respect to human glmerulonephritis, many experimentalmodels have been reported, most of them are those about acuteglomerulonephritis and have only a similarity in morphologic changes butno similarity in clinical functions such as metabolism, etc. Therefore,they cannot be utilized for the developments of diagnosis andtherapeutic agents.

It is well known that there is Masugi's glomerulonephritis as one of theexperimental models. Masugi's glomerulonephritis is such phenomenon thatglomerulonephritis is induced accompanying with proteinuria by injectinga serum which is obtainable from steps of (1) injecting a homogenate ofkidney excised from normal animal to heterologous animal in the rate ofone or two times a week for about 2 months, of (2) collecting blood fromthe animal and of (3) separating the serum from the blood, to a normalanimal homologous to the animal from which kidney is excised. Asapparent from the course of inducing glomerulonephritis, it has beenbelieved that Masugi's glomerulonephritis is induced throughanitgen-antibody reaction using the homogenate of kidney.

Thereafter, many attempts have been made for purifying the homogerate toisolate a complete antigen substance that prepares "nephrotoxicantiserum" having an ability to induce glomerulonephritis. As theresults of anatomical attempts, it has been found that the completeantigen substance is mainly present in glomerulus of kidney, especiallyglomerular basement membrane (GBM). Although many attempts from thepoint of chemistry have been made, the complete antigen substance hasnot been made clear yet.

Spiro reported that two glycopeptides can be isolated from bovine GBM[Spiro, R. G., "J. Biol. Chem.", Vol. 242, No. 8, 1923-1932 (1967)].

Spiro's procedure for isolating the two glycopeptides is as follows.Firstly, GBM is separated from kidney according to Krakower andGreenspon's method [Krakower and Greenspon "Arch. Path" 51, 629-639,(1951)]. The separated GBM is dispersed in 0.1 M Tris acetate buffer anddigested with collagenase at pH of 7.4 and temperature of 37° C. Thesupernatant liquid obtainable from centrifuging the digestion solutionis conditioned at pH 7.8 and then is digested with pronase at 37° C.Thus digestion solution is centrifuged to collect the supernatantliquid.

The supernatant liquid thus obtained is fractionated with Sephadex G-25.As shown in FIG. 1 of the literature mentioned above, two fractionswhich have sugar peaks 1 and 2 (by anthrone) respectively are obtainedby this fractionation. The fraction (sugard peak 1) belonging to thevoid volume of Sephadex G-25 is further fractionated with Sephadex G-50.As shown in FIG. 5 of the literature mentioned above, sugar peak 1 isdivided into two sugar peaks (by anthrone), one of them belonging to thevoid volume of Sephadex G-50.

Spiro found from this procedure two glycopeptides from the sugar peak 2in FIG. 1 and the right sugar peak in FIG. 5. The glycopeptide of thesugar peak 2 is disaccharide comprising glucose and galactose in theratio of 1:1 and one molecule of hydroxylysin, while the glycopeptide ofthe another sugar peak is heteropolysaccharide comprising galactose,mannose, glucosamine and fucose. However, both of them have nobiological activity.

Spiro discarded the left sugar peak in FIG. 5 as impurities according tothe common knowledge in this field because it belongs to the voidvolume.

The inventor has studied an establishment of an experimental model forhuman glomerulonephritis, especially for adult human glomerulonephritis.

It was found surprisingly that there is contained a substance having anability to induce glomerulonephritis in animal in the left sugar peak inFIG. 5 which has been discarded by Spiro and that this substance isdifferent quite from the two glycopeptides isolated by Spiro. It wasfound also that the biological activity of glycopeptide of the presentinvention is displayed by injecting the glycopeptide directly to ananimal.

The inventor termed such biological activity of the glycopeptide of thepresent invention "nephrotogenicity" against that the biologicalactivity in Masugi's glomerulonephritis was termed "nephrotoxicity".

STATEMENT OF OBJECTS

It is an object of the present invention to provide a novel glycopeptideconsisting of a sugar moiety having a stractural formula ##STR3## and apeptide moiety linking to the sugar moiety through N-glycoside bond.

It is another object of the present invention to provide a process ofisolating the glycopeptide, comprising steps of (1) digesting an organof normal animal with a protein-digesting enzyme and centrifuging, of(2) digesting the supernatant liquid obtained in the step (1) withpronase and centrifuging, of (3) dialyzing the supernatant liquidobtained in the step (2), of (4) treating the non-dialyzable substancewith trichloroacetic acid and centrifuging, and of (5) contacting thesupernatant liquid obtained in the step (4) with concanavalin A andeluting the substance trapped on concanavalin A.

It is another object of the present invention to provide a process ofisolating the glycopeptide, comprising steps of (1) digesting an organof normal animal with a protein-digesting enzyme and centrifuging, of(2) digesting the supernatant liquid obtained in the step (1) withpronase and centrifuging, of (3) dialyzing the supernatant liquidobtained in the step (2), of (4) treating the non-dialyzable substancewith trichloroacetic acid and centrifuging, and of (5) contacting thesupernatant liquid obtained in the step (4) with non-water solublematerial having negative charge and eluting the substance adsorbed onthe non-water soluble material.

It is another object of the present invention to provide a process ofisolating the glycopeptide comprising steps of (1) contacting urine fromhealthy human being or the concentrate thereof with non-water solublematerial having negative charge and eluting substances adsorbed on thenon-water soluble material, of (2) digesting the eluate with pronase andcentrifuging, of (3) dialyzing the supernatant liquid obtained in thestep (2), and of (4) treating the non-dialyzable substance withtrichloroacetic acid.

It is another object of the present invention to provide an agent ofinducing glomerulonephritis in an animal characterized in that itcontains the glycopeptide as an active component.

It is still another object of the present invention to provide a methodof preparing an experimental model useful for study on humanglomerulonephritis characterized in that the glycopeptide is injected toan experimental animal to induce lesions similar to those of humanglomerulonephritis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a gas-chromatographic chart of a product obtainable frommethylation of the glycopeptide of the present invention;

FIG. 2 is a ¹³ C NMR spectrum of the glycopeptide of the presentinvention;

FIG. 3 is a microscopic photography showing renal tissue of rat after 8months from an injection of the glycopeptide; and

FIG. 4 is a microscopic photography showing renal tissue of normal rat.

DETAILED DESCRIPTION OF INVENTION

The glycopeptide of the present invention consists of a sugar moietyhaving a structural formula ##STR4## and a peptide moiety linking to thesugar moiety through N-glycoside bond.

This glycopeptide is characterized not only by novel structure of thesugar moiety but also by unique property. In other words, while Masugi'sglomerulonephritis is induced through the antigen-antibody reaction,glomerulonephritis in the case of the present invention, is induced by asingle injection in hind footpad of the animal and the inducedglomerulonephritis makes progress to chronic glomerulonephritis(contracted kidney) after 6-8 months from the injection.

The nephritogenicity of the glycopeptide is derived from the sugarmoiety. More detailedly, it was confirmed that although even if thepeptide moiety is destroyed by suitable means such as digestion withtrypsin or collagenase and pronase, the nephritogenicity is not damagedat all, when the sugar moiety is destroyed by IO₄ oxidation,glomerulonephritis is not induced at all.

Now, the determination of structure of the sugar moiety which controlsthe nephritogenicity will be detailed hereinafter.

i. consisting of glucose only

According to usual method in the determination of structure of sugar[Hakomori, S. J. Biochem. 55, 205-208, (1964)], the glycopeptide sampleisolated from rat GBM was methylated and the methylated product wassubjected to gas-chromatography. FIG. 1 shows the obtainedgas-chromatographic spectrum. The compounds in the peaks of the spectrumwere identified by Mass spectrometory. The obtained results werereported in Table.

                  TABLE                                                           ______________________________________                                        peak       compound                                                           ______________________________________                                        A          2,3,4,6-tetra-O--methyl-D-glucose                                  B          3,4,6-tri-O--methyl-D-glucose                                      C          2,3,6- and 2,3,4-tri-O--methyl-D-glucose                           D          3,6-di-O--methyl-D-glucose                                         E          2,4-di-O--methyl-D-glucose                                         ______________________________________                                    

As the results, it was found that the sugar moiety is composed ofglucose residue only.

ii. Non-reducing terminus of sugar moiety being α-D-glucose

It was found that the glycopeptide of the present invention has acharacteristic of bonding to concanavalin A (Con A). It is well knownthe Con A has a property of reacting selectively with polysaccharidewhich has any of α-D-glucose, α-D-mannose, orβ-D-fructofuranosyl unit inits non-reducing terminus so as to produce precipitates. In view of thisfact and the results of the item i, it was decided that the glucose innon-reducing terminus of the sugar moiety is α-D-type.

iii. sugar moiety consisting of three glucose residues

The glycopeptide was analysed by ¹³ C NMR spectrometory using t-butylalcohol as internal standard (solvent: D₂ O). FIG. 2 shows ¹³ C NMRspectrum thus obtained.

According to J. B. STOTHERS, "Carbon-13 NMR Spectroscopy" AcademicPress, New York, (1972), Chap. 11 and E. BREITMAIER and W. VOELTER, "¹³C NMR Spectroscopy", Methods and Applications, Verlag Chemic Weinheim,Germany, (1974), Chap. 5, it is well known that the chemical shift rangeof carbon C(1) in glucose and its analogues, which is bonded to twooxygen atoms, is 110-90 PPM (PPM from TMS) and that of carbons C(2)-C(6)which are bonded to one oxygen atom, is 85-60 PPM.

It is evident from the studies of the intensities of the signals in theregion of 110-60 PPM that the number of carbon of the sugar moiety isless than 24. This indicates that the number of sugar residue in thesugar moiety is 3 or 4, because one glucose ring has six carbon atomsand there may be some possibilities that the signals of other carbons inthe peptide moiety of the glycopeptide are in this region.

The glycopeptide has only two peaks A (104.2 PPM) and B (100.2 PPM) inthe region of the chemical shifts of carbon (1) in glucose ring. Theintensities of these two signals are almost the same with each other.This fact eliminates the possibility of the presence of four glucoseresidues in the glycopeptide. Accordingly, it is evident that the sugarmoiety is composed of three glucose residues.

iv. sugar moiety having bond pattern of gluα-1.6 gluβ-1.6 gluα

In order to assign the signals of carbons in the sugar moiety of theglycopeptide, ¹³ C NMR spectra of 27 kinds of analogous sugars and theirderivatives were measured at almost the same condition. From theanalyses of these ¹³ C NMR spectra, it has become evident that thesignal B (100.2 PPM) (see FIG. 2) can be assigned to the carbon (1) ofα-1,6 glucose-glucose linkage, because the observed chemical shift ofisomaltose (α-1,6 linkage) (100.5 PPM) is very close to that of B.However, the signal A (104.2 PPM) cannot be immediately assigned,because any peaks of analogous sugars are not so close to that of A. Asthe chemical shifts of one glucose ring are influenced by the type oflinkage of the second and/or the third glucose ring, substituent effectsby the glucose ring on the chemical shifts were obtained by the detailanalyses of the chemical shifts of glucose-glucose chain compounds withα-, β-1,1-, 1,2-, 1,3-, 1,4-, and 1,6-linkages. Using the values of thesubstituent effects and the chemical shifts of glucose thus obtained,expected chemical shifts of many types of combinations of three glucoserings were calculated. Thus, following five types were chosen as theapparopriate combinations to fit the two peaks A and B or theglycopeptide from the analyses of C(1) chemical shifts. ##STR5##

In the analyses of the chemical shift range of 85-60 PPM, it is expectedthat the chemical shift of the bonded carbon at 4-position of glucose ofβ-1,4 linkage must be near 81.4-81.2, considering the experimentallyobtained value of cellobiose (β-1,4 linkage) (81.2 PPM) and the resultsof the calculations. However, such a signal cannot be found in thespectrum of the glycopeptide. The chemical shift of its nearest peak is77.7 PPM, but it is too far. Therefore, types III, IV and V areexcluded.

Moreover, from the Concanavalin A test, it has been made clear that thenon-reducing terminus glucose in the glycopeptide has an α-configration.This and ¹³ C NMR analyses indicate that the sugar moirty hasα-1,6-β-1,6-α chain configration.

v. peptide moiety linking to sugar moiety through N-glycoside bond

According to the method of hydrolysis with strong alkali which isutilized ordinarily for destroying N-glycoside bond between asparagineor glutamine and sugar [Lee, Y. C. and Scocca, J. R. "J. Biol. Chem."247, 5753-5758, (1972)], the glycopeptide of the present invention washydrolized. Most of the products were trisaccharide (this correspondswith the fact described in the item iii).

Moreover, with respect to ¹³ C NMR, a methine carbon which is bonded toone oxygen atom and one nitrogen atom (--O--CH--N<) is expected toresonate at much higher field (probably in the range of 85-70 PPM) thanthat bonded to two oxygen atoms. Taking this and only two signals withthe same intensities for C(1) into account, it can be said that thesugar moiety has an N-glycoside linkage at C(1) of the terminal glucose,instead of the peptide linkage through a glucosamine.

From these facts, it is clear that the sugar moiety links to the peptidemoiety through N-glycoside bond.

In view of the facts mentioned in the items i-v, it was concluded thatthe structural formula of the sugar moiety is as follows. ##STR6##

Now, the process of isolating the glycopeptide will be detailedhereinafter.

The GBM prepared according to Krakower and Greenspon's method isdigested with a protein-digesting enzyme such as trypsin or collagenaseand centrifuged. The obtained supernatant liquid is digested withpronase, and is centrifuged. The obtained supernatant liquid is dialyzedto remove dialyzable disaccharide (corresponding to Spiro's sugar peak2). The remaining non-dialyzable solution is lyophilized. Thus obtainedpowder is dissolved to water and trichloroacetic acid is added to thesolution to form precipitates which are removed by centrifuging. Sincetrichloroacetic acid has a characteristic of removing selectivelyglycopeptide containing mannose as sugar component as well as well knowncharacteristic of reacting with free protein to form precipitates,Spiro's heteropolysaccharide (corresponding to the right sugar peak inFIG. 5 mentioned above) can be removed. Thereafter, the supernatantliquid is subjected to Con A affinity chromatography. As mentionedabove, Con A has the characteristic of bonding to α-D-glucose innon-reducing terminus of polysaccharide. Accordingly, the glycopeptideof the present invention is trapped on Con A. Then the glycopeptidetrapped on Con A is eluted with α-D-methyl mannose.

According to the inventor's study, since the glycopeptide has suchcharacteristic that it is adsorbed selectively on non-water solublematerial having negative charge, it is possible to utilize a step ofcontacting the supernatant liquid with the non-water soluble materialand eluting the substance adsorbed on the non-water soluble material, instead of the step of Con A affinity chromatography.

As the non-water soluble material having negative charge, it is able touse polymeric materials containing functional group having negativecharge or inorganic materials having negative charge. The typicalexamples of the former are the materials having nitrile group, carboxylgroup, sulfonic acid group or halogen atom, prepferably polyvinylchloride, polyacrylonitrile, cellulose and cotton. The typical examplesof the latter are porous glass, zeolite, silica gel, and sellite.

The optimum condition in the step of adsorbing is depend on the type ofnon-water soluble material, especially it is preferable to carry out atpH ranging from neutral to slight acidic.

As eluting solvent, it is able to use weak alkaline aqueous solutionsuch as diluted ammonia aqueous solution, or aqueous solution ofelectrolyte such as sodium chloride or an agent for modifying proteinsuch as urea.

According to the inventor's study, since the glycopeptide is presentalso in organs other than kidney, such as lung, aorta, liver, spleen,heart and muscle, it is possible to use these organs as a startingmaterial.

According to the inventor's another study, since the glycopeptide ispresent in urine from human being, it is able to isolate theglycopeptide starting from urine from healthy males preferably.

In this case, the urine is firstly contacted with non-water solublematerial. The adsorbed substance is eluted after washing of thenon-water soluble material with distilled water. It is able to carry outthe adsorption with good efficiency by concentrating previously theurine according to usual method, such as, for example, concentrationunder reduced pressure, concentration with ultrafiltration or foamingconcentration.

It is preferable to regulate the urine at pH ranging from neutral toslight acidic. In this case, it is also able to use as eluting solventweak alkaline aqueous solution or aqueous solution containingelectrolyte or agent for modifying protein.

Thus obtained eluate is digested with pronase and is centrifuged. Thesupernatant liquid is dialyzed. The remaining non-dialyzable substanceis treated with trichloroacetic acid and the produced precipitates areremoved by centrifuging.

If necessary, the obtained supernatant liquid is further subjected toCon A affinity chromatography and the substance trapped on the Con A iseluted with an eluent.

The nephritogenicity of the glycopeptide will be described hereinafter.

In animals which received a single footpad injection of the glycopeptidewith Freund's incomplete adjuvant, proteinuria began to appear 3-4 weeksafter the injection and increased gradually. Morphologic changes ofproliferative glomerulonephritis appeared 1.5 months after the injectionand progressed, 6 to 8 months after injection, to typical chronicglomerulonephritis (the contracted kidney). FIG. 3 shows lesional organof kidney after 8 months from the injection. It is found from thecomparison with normal kidney (shown in FIG. 4) that the morphologicchanges in kidney have progressed to typical chronic glomerulonephritis.

Furthermore, as the result of disorders of clinical metabolism, uniqueuremic osteodystrophy (a bone disease secondary of chronic renalfailure) was produced in animals: that is, severe osteodystrophy wasinduced, 300-400 days after the footpad injection in these animals, withmarked (over 10 fold) hyperplasia of the parathyroid gland and thedecrease of serum Ca level.

The reproducing efficiency of nephritogenicity of the glycopeptide isalmost 100%.

The present invention will be understood more readily by referenced tothe following examples; these examples are intended to illustrate theinvention and are not to be constructed to limit the scope of theinvention.

Some of the examples relate to the isolation using organs excised fromrat. However, it is possible, of course, to use equally as startingmaterial the organs excised from animals other than rat, such as, forexample, bovine, dog, rabbit, mouse, etc.

EXAMPLE 1 Isolation from rat GBM

Kidney excised from 1200 rats was washed fully with physiological saltsolution under reflux to remove blood and thereafter was cut finely tocollect renal cortex [Yield: about 1 kg (wet weight)].

The GBM was prepared from the renal cortex according to Krakower andGreenspon's method. More detailedly, the renal cortex was cut finely andseparated with sieves having 150 mesh and 100 mesh and finally 170 mesh.The glomerulus remaining on the sieve having 170 mesh was washed fullywith physiological salt solution and was subjected to treatment withsuper sonic wave. After washing with physiological salt solution manytimes and then with distilled water, the solution containing destroyedglomerulus was centrifuged at low speed. Pure GBM 20 g (wet weight) wasobtained as precipitate.

The GBM was dissolved to small amount of phisiological salt solutionadjusted to pH 8.0 with 0.1 M sodium borate. Trypsin was added in theamount of 0.5% of the weight of GBM and the mixture was incubated at 37°C. for 3 hours. The mixture was heated at 60° C. for 30 min. toinactivate trypsin and was centrifuged at 27,000 rpm for 35 min. Thesupernatant liquid was lyophilized.

The powder was dissolved to small amount of physiological salt solutionand the mixture was adjusted to pH 7.8 with Tris acetate buffer and wasdigested with pronase. This enzyme was added initially in the amount of0.3% with further enzyme additions of 0.1% at 24 and 48 hours. Thisincubation was carried out for a total period of 72 hours at 37° C.Thereafter, the undigested material was removed by centrifugation andthe obtained supernatant liquid was lyophilized.

The powder was dissolved to distilled water. The solution was chargedinto cellulose tube (Visking tube, 27/32 inch) and the cellulose tubewas dipped into the flowing water. The dialysis was continued for 3days. In this way, dialyzable disaccharide was removed.

Trichloroacetic acid was then added to the nondialyzable materials inthe amount of 5% and the mixture was stood still for short time. Theproduced precipitates were removed by centrifuging at 3,000 rpm for 30min., whereby heteropolysaccharide was removed.

The obtained supernatant liquid was fed to a column fulfilled with ConA. After washing the column with distilled water, the substance trappedon the Con A was eluted with α-D-methyl mannose. The eluate waslyophilized so as to obtain the glycopeptide (10 mg).

It was confirmed by ¹³ C NMR analysis that the glycopeptide obtained bythe process mentioned above has two signals at 104.2 PPM and 100.2 PPM.

EXAMPLE 2 Isolation from rat renal cortex

The renal cortex about 1 kg (wet weight) treated similarly to Example 1was separated with a sieve having 9 mesh. The renal cortex which passedthrough the sieve was washed with small amount of physiological saltsolution and then was dialyzed and lyophilized after desalting. Thepowder was dissolved to water and adjusted at pH 8.0 with 0.1 M sodiumborate.

The preparation of the glycopeptide was carried out using this solutionas a starting material similarly to Example 1. The glycopeptide wasobtained with yield of 6-8 mg.

EXAMPLE 3 Isolation from rat lung

The lung excised from rat was washed fully with physiological saltsolution under reflux in order to remove blood. The lung was cut finelyand put into acetone. The lung tissue precipitating in acetone wascollected and dried after removing acetone. Then, the powder wasseparated with a sieve having 9 mesh to remove bronchus.

Thus obtained lung tissue 200 g (dry weight) was used as a startingmaterial. The preparation was carried out similarly to the Example 1.The glycopeptide was obtained with yield of 10 mg.

EXAMPLE 4 Isolation from rat aorta

The aorta excised from rat was put into acetone. After some days,acetone was removed and the aorta was dried and was milled with amortar. Thus obtained aorta tissue 120 g was used as a startingmaterial.

The preparation of glycopeptide from aorta tissue was carried outsimilarly to the Example 1. The glycopeptide was obtained with yield of10 mg.

EXAMPLE 5 Isolation from rat liver

The liver excised from rat was washed fully with physiological saltsolution under reflux to remove blood and then homogenized. Thehomogenate was centrifuged at 8,000 rpm to collect precipitates.

The isolation of glycopeptide was carried out using the precipitates1,000 g (wet weight) similarly to the Example 1. The glycopeptide wasobtained with yield of 5 mg.

EXAMPLE 6 Isolation from rat spleen

Using spleen excised from rat, the starting material was preparedsimilarly to the Example 5.

The isolation of glycopeptide from the starting material 1,000 g (wetweight) was carried out similarly to the Example 1. The glycopeptide wasobtained with yield of 2-3 mg.

EXAMPLE 7 Isolation from rat heart

In this case, the homogenate prepared by homogenizing heart excised fromrat was used as a starting material.

Using the homogenate 1,000 g, the isolation of glycopeptide was carriedout similarly to the Example 1. The glycopeptide was obtained with yieldof 6 mg.

EXAMPLE 8 Isolation from rat muscle

Using rat muscle, the starting material was prepared similarly to theExample 5.

The isolation of glycopeptide from this starting material 1 kg wascarried out simialrly to the Example 1. The glycopeptide was obtainedwith yield of 2 mg.

EXAMPLE 9

The preparation of glycopeptide from rat GBM was carried out as shown inthe Example 1 but, in stead of the step of Con A affinitychromatography, the step of adsorption with non-water soluble materialwas utilized in order to extract selectively the glycopeptide. Moredetailedly, the supernatant liquid obtained after the treatment withtrichloroacetic acid was fed to the column fulfilled with sellaite.After washing the sellaite with distilled water, the glycopeptideadsorbed on the sellaite was eluted with ammonia aqueous solution. Theeluate was neutralized and lyophilized after desalting. Whereby, theglycopeptide was obtained with yield of 10 mg.

It was confirmed by ¹³ C NMR analysis that the obtained glycopeptide hastwo signals at 104 PPM and 100 PPM.

In the operation mentioned above, even if polyacrylonitrile fiber,polyvinyl chloride, cellulose, cotton, porous glass, zeolite, or silicagel is used in stead of sellaite, it is able to obtain equal results.

EXAMPLE 10 Isolation from human urine

Urine 1,000 l from healthy males was concentrated by feeding air intourine for 2 hours and then adding small amount of octanol to theproduced foam. In this way, the concentrated urine 100 l was obtained.The concentrated urine was diluted with distilled water 100 l and wasadjusted to pH 6.5. To this solution, sellaite 100 l was added and themixture was agitated for 1 hour. Thereafter, the sellaite was collectedby filtration. The separated sellaite was washed with alkaline water.The substance adsorbed on the sellaite was then eluted with alkalinesolution containing sodium chloride. The eluate was neutralized andlyophilized after desalting with superfiltration and concentrating. Thedry urine powder 2 g was obtained.

The powder was dissolved to small of physiological salt solution and themixture was adjusted to pH 7.8 with Tris acetate buffer and digestedwith pronase. This enzyme was added initially in the amount of 0.3% andfurther in the amount of 0.1% at 24 and 48 hours. This incubation wascarried out for a total period of 72 hours at 37° C. Thereafter, theundigested material was removed by centrifugation and the obtainedsupernatant liquid was lyophilized.

The powder was dissolved to distilled water. The solution was chargedinto cellulose tube (Visking tube, 27/32 inch) and the cellulose tubewas dipped into the flowing water. The dialysis was continued for 3days. In this way the dialyzable disaccharide was removed.

Trichloroacetic acid was added to the remaining non-dialyzable materialsin the amount of 5% and the mixture was stood still for short time. Theproduced precipitates were removed by centrifuging at 3,000 rpm for 30min. The obtained supernatant liquid was lyophilized (yield of 10-15mg).

In order to obtain the glycopeptide having higher purity, thesupernatant liquid after the treatment with trichloroacetic acid was fedto a column fulfilled with Con A. After the column was washed withdistilled water, the substance trapped on the Con A was eluted withα-D-methyl mannose. The eluate was lyophilized.

It was confirmed by ¹³ C NMR analysis that the glycopeptide from humanurine also has two signals at 104 PPM and 100 PPM.

EXAMPLE 11

The concentrated urine 100 l obtained by the treatment same to that inthe Example 10 was adjusted to pH 5.5 and then fed gradually to a columnfulfilled with polyacrylonitrile fiber 300 g. After the polyacrylnitrilefiber was washed fully with water, the substance adsorbed thereon waseluted with 4% ammonia aqueous solution. The eluate was neutralized andtreated similarly to the Example 10 so as to obtain dry urine powder 2g.

The isolation of glycopeptide from the urine powder was carried outsimilarly to the Example 10. The glycopeptide was obtained with yield of10-15 mg.

Example 12

Using polyvinyl chloride in stead of polyacrylonitrile fiber, the dryurine powder 2 g was obtained similarly to the Example 11.

The isolation of glycopeptide from the dry urine powder was carried outsimilarly to the Example 10 (Yield: 10-15 mg).

EXAMPLE 13

Human urine 1,000 l was adjusted to pH 7.5. After the producedprecipitates were removed, the urine was fed at speed of 500 cc/min. tothe column fulfiled with silica gel 30 l which was previously washedwith 5% HCl and 10% NaCl aqueous solution. Thereafter, the column waswahsed with water and the substance adsorbed on the silica gel waseluted with 4% ammonia aqueous solution 4 l. The eluate was neutralizedwith HCl and was concentrated and finally lyophilized so as to obtaindry urine powder 4 g.

The isolation of glycopeptide from dry urine powder was carried outsimilarly to the Example 10 (Yield: 20-25 mg).

EXAMPLE 14

Human urine 1,000 l was fed at speed of 200 l/hour to a column (diameter6 cm; height 30 cm) fulfilled with porous glass 1 l having mesh size of120-200 mesh. Thereafter, the column was washed with water 10 l and thesubstance adsorbed on porous glass was eluted with 4% ammonia aqueoussolution 4 l. The eluate was neutralized with 5% HCl and wasconcentrated and finally lyophilized so as to obtain dry urine powder4.5 g.

The isolation of glycopeptide from the dry urine powder was carried outsimialrly to the Example 10 (Yield: 20-25 mg).

The nephritogenicity of glycopeptide obtained in the examples mentionedabove will be illustrated by the following experiments.

EXPERIMENT 1

The glycopeptide isolated from rat GBM (Example 1) was injected tofootpad of rat with Freund's incomplete adjuvant. The injection amountof glycopeptide was 300-500 μg. After 8 months from the injection therat was killed and dissected. It was confirmed that morphologic changesof kidney progressed to the contracted kidney which is a typical changeof chronic glomerulonephritis.

EXPERIMENT 2

The glycopeptide (300-500 μg) isolated from rat lung (Example 3) wasinjected to footpad of rat. After 8 months from the injection, it wasconfirmed that morphologic changes of kidney progressed to thecontracted kidney.

When the glycopeptide isolated from other organs, that is, aorta, liver,spleen, heart or muscle was injected to rat, the morphologic changeswere the same to those observed in the case of GBM or lung.

EXPERIMENT 3

The glycopeptide (300-500 μg) isolated from human urine (Example 10) wasinjected to footpad of rat. After 8 months from the injection, it wasobserved that morphologic changes of kidney progressed to the contractedkidney.

EXPERIMENT 4

The glycopeptide (300-500 μg) isolated from rat GBM was injected to dogwith Freund's incomplete adjuvant. Similarly to the case of rat, after 8months from the injection, the morphologic changes of kidney progressedto the contracted kidney.

While the described embodiment represents the preferred form of thepresent invention, it is to be distinctly understood that the inventionis not limited thereto but may be otherwise variously embodied withinthe scope of the following claims.

What is claimed is:
 1. A process of isolating a glycopeptide consistingof a sugar moiety having a structural formula ##STR7## and a peptidemoiety linking to the sugar moiety through N-glycoside bond, comprisingsteps of (1) digesting an organ of normal animal with aprotein-digesting enzyme and centrifuging, of (2) digesting thesupernatant liquid obtained in the step (1) with pronase andcentrifuging, of (3) dialyzing the supernatant liquid obtained in thestep (2), of (4) treating the non-dialyzable substance withtrichloroacetic acid and centrifuging, and of (5) contacting thesupernatant liquid obtained in the step (4) with non-water solublematerial having negative charge and eluting the substance adsorbed onthe non-water soluble material,wherein said peptide moiety is oneobtained from said organ of normal animal which is selected from thegroup consisting of kidney, lung, aorta, liver, spleen, heart andmuscle.
 2. The process claimed in the claim 1 wherein the non-watersoluble material having negative charge is a polymeric materialcontaining a functional group having negative charge, or an inorganicmaterial having negative charge.
 3. The process claimed in the claim 2wherein the polymeric material is the one containing nitrile group,carboxyl group, sulfonic acid group or halogen atom.
 4. The processclaimed in the claim 3 wherein the polymeric material is polyvinylchloride, polyacrylonitrile, cellulose, or cotton.
 5. The processclaimed in the claim 2 wherein the inorganic material having negativecharge is porous glass, zeolite, silica gel or sellaite.
 6. A process ofisolating a glycopeptide consisting of a sugar moiety having astructural formula ##STR8## and a peptide moiety linking to the sugarmoiety through N-glycoside bond, comprising steps of (1) contactingurine from healthy human being or the concentrate thereof with non-watersoluble material having negative charge and eluting substance absorbedon the non-water soluble material, of (2) digesting the eluate withpronase and centrifuging, of (3) dialyzing the supernatant liquidobtained in the step (3), of (4) treating the non-dialyzable substancewith trichloroacetic acid and (5) separating the supernatant containingglycopeptide from the precipitate formed in step (4).